Showing papers by "Fabien Zoulim published in 2009"

Persistence of the hepatitis B virus covalently closed circular DNA in HepaRG human hepatocyte-like cells.

Abstract: The recently described hepatic cell line HepaRG is the sole hepatoma cell line susceptible to hepatitis B virus (HBV) infection. It provides a unique tool for investigating some unresolved issues of the virus' biology, particularly the formation of the viral mini-chromosome believed to be responsible for the persistence of infection. In this study, we characterized the main features of HBV infection: it is restricted to a subpopulation of differentiated hepatocyte-like cells that express albumin as a functional marker and represents around 10 % of all differentiated HepaRG cells. Infection may persist for more than 100 days in cells maintained at the differentiated state. Even though infected cells continued to produce infectious viral particles, very limited or no spreading of infection was observed. Low genetic variation was also observed in the viral DNA from viruses found in the supernatant of infected cells, although this cannot explain the lack of reinfection. HBV infection of HepaRG cells appears to be a very slow process: viral replication starts at around day 8 post-infection and reaches a maximum at day 13. Analysis of viral DNA showed slow and inefficient conversion of the input relaxed circular DNA into covalently closed circular (CCC) DNA, but no further amplification. Continuous lamivudine treatment inhibited viral replication, but neither prevented viral infection nor initial formation of CCC DNA. In conclusion, HBV infection in differentiated HepaRG cells is characterized by long-term persistence without a key feature of hepadnaviruses, the so-called 'CCC DNA amplification' described in the duck hepatitis B model.

Receptor complementation and mutagenesis reveal SR-BI as an essential HCV entry factor and functionally imply its intra- and extra-cellular domains.

Abstract: HCV entry into cells is a multi-step and slow process. It is believed that the initial capture of HCV particles by glycosaminoglycans and/or lipoprotein receptors is followed by coordinated interactions with the scavenger receptor class B type I (SR-BI), a major receptor of high-density lipoprotein (HDL), the CD81 tetraspanin, and the tight junction protein Claudin-1, ultimately leading to uptake and cellular penetration of HCV via low-pH endosomes. Several reports have indicated that HDL promotes HCV entry through interaction with SR-BI. This pathway remains largely elusive, although it was shown that HDL neither associates with HCV particles nor modulates HCV binding to SR-BI. In contrast to CD81 and Claudin-1, the importance of SR-BI has only been addressed indirectly because of lack of cells in which functional complementation assays with mutant receptors could be performed. Here we identified for the first time two cell types that supported HCVpp and HCVcc entry upon ectopic SR-BI expression. Remarkably, the undetectable expression of SR-BI in rat hepatoma cells allowed unambiguous investigation of human SR-BI functions during HCV entry. By expressing different SR-BI mutants in either cell line, our results revealed features of SR-BI intracellular domains that influence HCV infectivity without affecting receptor binding and stimulation of HCV entry induced by HDL/SR-BI interaction. Conversely, we identified positions of SR-BI ectodomain that, by altering HCV binding, inhibit entry. Finally, we characterized alternative ectodomain determinants that, by reducing SR-BI cholesterol uptake and efflux functions, abolish HDL-mediated infection-enhancement. Altogether, we demonstrate that SR-BI is an essential HCV entry factor. Moreover, our results highlight specific SR-BI determinants required during HCV entry and physiological lipid transfer functions hijacked by HCV to favor infection.

Altered expression profiles of microRNAs in a stable hepatitis B virus-expressing cell line.

Abstract: Background MicroRNAs (miRNAs) are highly conserved small non-coding RNAs of 18 - 25 nucleotides (nt) that mediate post-transcriptional gene regulation. Hepatitis B virus (HBV) can cause either acute or chronic hepatitis B, and is a high risk factor for liver cirrhosis and hepatocellular carcinoma. Some mammalian viruses have been shown to modulate the expression of host cellular miRNAs. However, interactions between the HBV and the host cellular miRNAs are largely unknown. Methods miRNA microarray and Northern blotting analysis were used to compare the expression profile of cellular miRNAs of a stable HBV-expressing cell line HepG2.2.15 and its parent cell line HepG2. mRNA microarray assay and the miRanda program were used to predict the miRNA targets. A flow cytometric assay was further used to investigate the expression of human leukocyte antigen (HLA)-A. Results Eighteen miRNAs were differentially expressed between the two cell lines. Among them, eleven were up-regulated and seven were down-regulated in HepG2.2.15 cells. Northern blotting analysis confirmed that the expression of miR-181a, miR-181b, miR-200b and miR-146a were up-regulated and the expression of miR-15a was down-regulated, which was in consistent with the results of the microarray analysis. Furthermore, some putative miRNA targets were predicted and verified to be linked with mRNA expression. The 3'-UTR of HLA-A gene had one partially complementary site for miR-181a and miR-181a might down-regulate the expression of HLA-A. Conclusion HBV replication modulates the expression of host cellular miRNAs, which may play a role in the pathogenesis of HBV-related liver diseases.

Clearance of serum HBsAg and anti-HBs seroconversion following antiviral therapy for chronic hepatitis B.

Abstract: In this study, we have analyzed the evolution of serum HBsAg levels in 16 patients with chronic hepatitis B who showed an HBsAg seroconversion following antiviral therapy. The data showed that the clearance of serum HBsAg is slower than that of serum HBV DNA, which may reflect a slow kinetics of clearance of infected hepatocytes. Interestingly, HBsAg was detectable for a longer time using the Architect assay than with the Bio-Rad assay. As viremia suppression is achieved in most patients under therapy with the new generation of nucleoside analogs, these data suggest that the quantitative monitoring of serum HBsAg may represent a novel tool for the assessment of antiviral therapy efficacy.

Management and prevention of drug resistance in chronic hepatitis B.

Abstract: The management of hepatitis B virus resistance to antivirals has evolved rapidly in recent years. The definition of resistance is now well established, with the importance of partial response and the improvement of assays to detect genotypic resistance and virological breakthrough. Data on phenotypic resistance have allowed to define the cross-resistance profile for the main resistant mutants, providing a rationale for treatment adaptation. Clinical studies have shown that an early treatment intervention in case of a virological breakthrough or a partial response with the addition of a second drug having a complementary cross-resistance profile allows one to maintain the majority of patients in clinical remission. The prevention of resistance should rely on the use of the most potent antivirals with a high genetic barrier to resistance as a first-line therapy. The future perspectives are to design strategies to hasten the HBsAg clearance, which should become a new treatment endpoint, to prevent drug resistance and to decrease the incidence of complications of chronic hepatitis B.

Adefovir dipivoxil resistance patterns in patients with lamivudine-resistant chronic hepatitis B

Abstract: BackgroundLamivudine (3TC)-resistant chronic hepatitis B patients demonstrated a higher rate of adefovir dipivoxil (ADV) resistance compared with nucleoside-naive patients. This study describes ADV...

Inhibitory effect of the combination of CpG-induced cytokines with lamivudine against hepatitis B virus replication in vitro.

Abstract: BackgroundCurrently approved antiviral monotherapies against chronic hepatitis B fail to eradicate hepatitis B virus (HBV), to overcome the defects in HBV-specific immune responses and to prevent H...

Pathobiology of HBV mutants and clinical impact for treatment monitoring.

Abstract: HBV is the leading cause of liver cancer and frequently leads to cirrhosis and liver failure. The goals of nucleos(t)ide analog treatments are to suppress viral replication to as low as possible, to halt disease progression and to prevent the onset of complications. Due to the mechanism of viral genome persistence, chronic hepatitis B requires long-term therapy that exposes patients to the risk of selection of resistant mutants and treatment failure. Genotypic resistance is defined as the detection of resistant mutations that are known to confer resistance to antiviral drugs. Virologic rebound is defined as an increase in viral load of at least 1 log(10) copies/ml compared with the lowest value during therapy. Clinical breakthrough is defined as a rise in alanine aminotransferase levels and liver disease progression following the emergence of resistant mutants and the rise in viral load. Currently, the management of antiviral therapy should be based on precise virologic monitoring to enable an early diagnosis of partial response and treatment failure. An early treatment adaptation is recommended at least at the time of virologic breakthrough or in the case of insufficient viral suppression. The choice of drug for second-line therapy should be based on cross-resistance data to tailor therapy to the virologic situation of the patient. The addition of a complementary drug is the preferred strategy. Clinical experience shows that an optimal management of antiviral therapy allows the control of viral replication in the majority of patients in whom liver disease progression is halted.

Sexually transmitted HCV infection and reinfection in HIV-infected homosexual men.

Abstract: Summary Multiple, concomitant or successive hepatitis C virus (HCV) infections have been described in injection drug users and following organ transplantation and blood transfusion. However, data on sexual HCV reinfection is scarce. We report sexual HCV reinfection following viral eradication of a first HCV infection in two homosexual HIV-infected men. The first patient acquired HCV genotype 4 infection after resolution of an initial acute HCV genotype 1a infection. The second patient was infected with genotype 1a HCV following remission of an initial acute HCV genotype 4c/d infection. The two subjects were successfully treated with peginterferon alpha-2a and ribavirin for their first and second infection and achieved a sustained virological response on both occasions. Unprotected anal intercourse with multiple partners known to be HIV-positive (serosorting) was the only risk factor for HCV transmission reported by both patients. Therefore, sexual HCV reinfection can occur in homosexual men having unprotected sex and “serosorting” should be considered a risk factor for the sexual transmission of HCV.

Adefovir dipivoxil is effective for the treatment of cirrhotic patients with lamivudine failure.

Abstract: Background/Aims: Data on the efficacy of adefovir dipivoxil (ADV) in elderly and cirrhotic patients with lamivudine-resistant (LAM-R) chronic hepatitis B are scarce. This retrospective cohort study evaluated the safety and efficacy of ADV in this specific patient population. Methods: Sixty-eight cirrhotic LAM-R patients, of whom 19 (27.9%) were elderly (≥65 years of age) and nine had severe disease (two post-orthotopic liver transplantation, four pre-orthotopic liver transplantation and three decompensated), with hepatitis B virus (HBV) infection received ADV. Virological and biochemical responses to the addition of ADV were analysed. Results: At inclusion, all patients were receiving LAM; ADV was added. 75.4% of patients received a combination of LAM and ADV throughout this study for a median treatment duration of 12.6 months; the remainder received ADV with an overlap with LAM treatment for a median duration of 7.9 months. At the end of follow-up, 41.2% of patients had undetectable HBV DNA (≤2000 copies/ml) with a median reduction of 3.4 log10 copies/ml. Time to reach undetectable HBV DNA was dependent on baseline alanine aminotransferase (ALT) levels and HBeAg status. Normalization of serum ALT levels was observed in 55.2% (32/58) of patients. In patients who were HBeAg positive at baseline, HBeAg loss and seroconversion occurred in 23% (9/39) and 10% (4/39) respectively. No resistance mutations and no significant side effects were observed during the study period. Conclusion: Adefovir dipivoxil provides effective and safe treatment in cirrhotic and elderly patients who failed LAM therapy.

Inhibition of the binding of HCV serum particles to human hepatocytes by E1E2-specific D32.10 monoclonal antibody.

Abstract: The aim of this study was to determine the inhibition of binding activity of the monoclonal antibody (mAb) D32.10 which recognizes a highly conserved discontinuous antigenic determinant (E1:297-306, E2:480-494, and E2:613-621) expressed on the surface of serum-derived HCV particles (HCVsp) of genotypes 1a, 1b, 2a, and 3a. To this end, an in vitro direct cell-binding assay based on the attachment of radiolabeled HCVsp was developed, and Scatchard plots were used to analyze ligand-receptor binding data. HCV adsorption was also assessed by quantitating cell-associated viral RNA by a real-time RT-PCR method. Saturable concentration-dependent specific binding of HCVsp to Huh-7 or HepaRG cells was demonstrated. The Scatchard transformed data showed two-site interaction for Huh-7 and proliferative HepaRG cells: the high-affinity binding sites (K(d1) = 0.1-0.5 microg/ml) and the low-affinity binding sites (K(d1) = 5-10 microg/ml), and one-site high-affinity binding model between E1E2/D32.10-positive HCVsp and hepatocyte-like differentiated HepaRG cells. The E1E2-specific mAb D32.10 inhibited efficiently (>60%) and selectively the binding with an IC(50)

Traitement de l’hépatite B : progrès attendus

Abstract: Despite the development of new anitiviral agents, the treatment of chronic hepatitis B remains a major clinical challenge. Major achievements have been made with the rationale use of antivirals exhibiting a complementary cross resistance profile to prevent antiviral drug resistance. The current concept of modern antiviral therapy of chronic hepatitis B relies on a precise virologic monitoring and early treatment adaptation to prevent drug resistance. The difficulty of achieving viral clearance and the risk of drug resistance development are major arguments to continue research in the field of antivirals and to identify new targets for therapy. The development of true combination therapy is highly desirable to fulfil the objective of long-term viral suppression, clearance of viral cccDNA and infected cells and ultimately cure of the disease.

Clinical Aspects of Hepatitis C Virus Infection

Abstract: Recently, the most significant progress in knowledge of hepatitis C virus (HCV) biology and interaction with host cells has concerned the role of lipids for genome replication, assembly and egress, and entry into cells. The major hepatic consequence of HCV infection is the progression to cirrhosis and its complications, such as ascites, hepatic insufficiency, and hepatocellular carcinoma (HCC). With the identification in 1989 of HCV as the infectious agent responsible for non-A/non-B hepatitis and the development of specific detection tests, it has become possible to monitor the efficacy of interferon (IFN)-α treatment. With the increased survival associated with the use of highly active antiretroviral therapy (HAART), morbidity and mortality from HCV-induced liver disease have started to increase significantly. Microarray results from liver biopsy tissue taken before therapy in a cohort of patients given pegIFN and ribavirin were recently reported. In this study, patients who were subsequently identified as non-responders had high baseline expression of IFN-stimulated genes (ISGs), whereas responders to therapy more closely resembled healthy controls. Kempf et al. illustrated the need for good knowledge of drug metabolism to improve the efficacy of a drug in vivo. In this study it was shown that a pharmacokinetic enhancement of telaprevir and boceprevir could be achieved by codosing with ritonavir, a potent inhibitor of the cytochrome P450 3A, which is involved in the metabolism of both drugs.

P.111 Caractéristiques cliniques et histologiques, traitement et survie de 40 patients atteints de carcinome hépatocellulaire en l’absence de cirrhose

Abstract: Introduction Le carcinome hepatocellulaire (CHC) survient essentiellement sur foie cirrhotique. Dans 10 a 25 % des cas, il est cependant diagnostique en l’absence de cirrhose. Peu de donnees sont disponibles sur ce sujet dans la litterature. L’objectif de notre etude etait de preciser les caracteristiques epidemiologiques, le traitement et la survie de tels patients. Patients et Methodes Etude retrospective incluant tous les patients atteints de CHC, hospitalises dans notre institution (hepatologie et chirurgie viscerale) entre 1996 et 2007. Les criteres d’inclusion etaient un CHC prouve histologiquement developpe sur foie non fibreux ≤ F2 (selon le score METAVIR) avec relecture histologique centralisee. Les criteres d’exclusion etaient l’absence de donnees histologiques du CHC et/ou du foie adjacent, une fibrose hepatique > F2 et des signes radiologiques ou endoscopiques de cirrhose. Resultats 40/648 patients etaient inclus (6,2 %). La moyenne d’âge etait de 60,1 ans. Les facteurs de risque specifiques ou non du foie etaient : alcool > 30 g/j (10 cas) et/ou hepatites virales B et/ou C actives ou gueries (5 cas) et/ou hyperferritinemie (1 cas) et/ou adenomes hepatiques (4 cas) et/ou exposition medicamenteuse ou toxique (8 cas) et/ou syndrome metabolique (10 cas). A l’histologie, la tumeur etait tres bien ou bien differenciee pour 26 cas, avec presence d’emboles vasculaires dans 17 cas. La fibrose hepatique etait significativement plus elevee chez les patients avec syndrome metabolique vs hepatites virales vs consommation excessive d’alcool (respectivement 80 % F2 vs 66 % vs 60 %) (p ) (p Conclusion Dans notre serie, 25 % des carcinomes hepatocellulaire sur foie non cirrhotique etaient associes a un syndrome metabolique (isole ou associe a un autre facteur de risque), soulignant son potentiel role hepatocarcinogene avant le stade de fibrose severe. Le traitement etait chirurgical avec une survie semblant superieure a celle des patients atteints de CHC de meme taille sur foie cirrhotique, en raison probablement des bonnes fonctions hepatiques sous-jacentes. Ces donnees a confirmer pourrait conduire a une surveillance precoce des patients presentant un syndrome metabolique.

HEP-05-0036.R1 Susceptibility to Antivirals of an HBV Strain With Mutations Conferring Resistance to Both Lamivudine and Adefovir

Abstract: Mutations within the Hepatitis B virus (HBV) polymerase gene conferring drug -resistance are selected during prolonged lamivudine (3TC) or adefovir dipivoxil (ADV) treatment. As there is no other appro ved drug against HBV, treatments with 3TC or ADV are used either sequentially or in addition depending on treatment response or failure. Considering the use of de novo or add -on 3TC+ADV bitherapy, we investigated the possibility of emergence of an HBV stra in harboring polymerase mutations conferring resistance to both 3TC (rtL180M+M204V) and ADV (rtN236T). We constructed the L180M+M204V+N236T mutant and determined its replication capacity and its susceptibility to different nucleos(t)ide analogs in transien tly transfected hepatoma cell lines. The triple mutant replicates its genome in vitro , but less efficiently than either the wild -type (wt) HBV or L180M+M204V and N236T mutants. Phenotypic assays indicated that the L180M+M204V+N236T mutant is resistant to p yrimidine analogs (3TC, -FTC, E -L -FD4C, L -FMAU). Compared to wt HBV, this mutant displays a 6 fold decreased susceptibility to ADV and entecavir and a 4 fold decreased susceptibility to tenofovir. Interferon D inhibited equally the replication of wt and L1 80M+M204V+N236T HBV. In conclusion, the combination of rtL180M+M204V and rtN236T mutations impairs HBV replication and confers resistance to both 3TC and ADV in vitro . This suggests that the emergence of the triple mutant may be delayed and associated with viral resistance in patients treated with 3TC+ADV. However, other nucleos(t)ide analogs in development showed an antiviral activity against this multiresistant strain in vitro . This provides a rationale for the clinical evaluation of de novo combination t herapies. inserm-00133179, version 1 - 3 Nov 2009

Iconography : Sexually transmitted HCV infection and reinfection in HIV-infected homosexual men

Abstract: Multiple, concomitant or successive hepatitis C virus (HCV) infections have been described in injection drug users and following organ transplantation and blood transfusion. However, data on sexual HCV reinfection is scarce. We report sexual HCV reinfection following viral eradication of a first HCV infection in two homosexual HIV-infected men. The first patient acquired HCV genotype 4 infection after resolution of an initial acute HCV genotype 1a infection. The second patient was infected with genotype 1a HCV following remission of an initial acute HCV genotype 4c/d infection. The two subjects were successfully treated with peginterferon alpha-2a and ribavirin for their first and second infection and achieved a sustained virological response on both occasions. Unprotected anal intercourse with multiple partners known to be HIV-positive (serosorting) was the only risk factor for HCV transmission reported by both patients. Therefore, sexual HCV reinfection can occur in homosexual men having unprotected sex and "serosorting" should be considered a risk factor for the sexual transmission of HCV.